Graduate School of Systems Life Sciences, Kyushu University, Nishi-ku, Fukuoka, Japan.
Anal Biochem. 2012 May 15;424(2):130-6. doi: 10.1016/j.ab.2012.01.036. Epub 2012 Feb 17.
Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH(2)) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.
在这里，我们使用由带有荧光猝灭剂（BHQ3）的修饰硫酸软骨素 [CS(X)] 和带有阳离子肽底物（FKKQGSFAKKK-NH2）和近红外（NIR）荧光团（Cy5.5）的树枝状聚合物修饰的聚离子复合物（PIC）报道了在癌细胞裂解物中蛋白激酶 Cα（PKCα）活性的荧光检测。当聚合物 1 通过静电相互作用与 CS(X) 形成 PIC 时，NIR 荧光通过 Cy5.5 和 BHQ3 之间的Förster 共振能量转移（FRET）有效地猝灭。然而，当聚合物 1 中的侧链肽被 PKCα 磷酸化时，这种猝灭的荧光由于 PIC 的解离而恢复。当将 PKCα 添加到 PIC 分散体中时，观察到荧光强度显着增加，而其荧光增加则随着 PKCα 抑制剂的浓度依赖性抑制而受到抑制。此外，我们的 PIC 足够坚固，可以在未经纯化的癌细胞裂解物中测量 PKCα 活性。这种对 PKCα 有响应的 PIC 提供了一种简单、快速、灵敏和坚固的方法，可用于在粗细胞裂解物中检测 PKCα 活性，适用于药物筛选格式和使用粗细胞裂解物进行癌症诊断。
